Plant myb transcription factor homologs

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a Myb-related transcription factor. The invention also relates to the construction of a chimeric gene encoding all or a portion of the Myb-related transcription factor, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the Myb-related transcription factor in a transformed host cell.

This application claims the benefit of U.S. Provisional Application No.60/110,609, filed Dec. 2, 1998.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingMyb-related transcription factors in plants and seeds.

BACKGROUND OF THE INVENTION

Improvement of crop plants for a variety of traits, including diseaseand pest resistance, and grain quality improvements such as oil, starchor protein composition, can be achieved by introducing new or modifiedgenes (transgenes) into the plant genome. Transcriptional activation ofgenes, including transgenes, is in general controlled by the promoterthrough a complex set of protein/DNA and protein/protein interactions.Promoters can impart patterns of expression that are either constitutiveor limited to specific tissues or times during development.

Transcriptional activation is primarily mediated through transcriptionfactors that interact with enhancer and promoter elements. Binding oftranscription factors to such DNA elements constitutes a crucial step intranscriptional initiation. Each transcription factor binds to itsspecific binding sequence in a promoter and activates expression of thelinked coding region through interactions with coactivators and/orproteins that are a part of the transcription complex.

Several plant genes have been identified that appear to encodetranscription factors structurally related to the cMyb protooncogenefamily of mammals. Central to the similarities shared by these proteinsis the Myb repeat DNA-binding domain containing conserved tryptophanresidues at certain positions, and a helix-turn-helix-related domain.Generally, Myb-related proteins from plants contain two of theserepeats, R2 and R3 (Kranz et al. (1998) Plant J 16:263-276), thoughproteins having only one repeat have been identified (e.g., Feldbruggeet al. (1997) Plant J 11:1079-1093). These Myb-related genes appear toencode a large family of plant transcription factors that are involvedin a diversity of gene regulation. For example, plant Myb-related geneshave been shown to regulate anthrocyanin biosynthesis in maize andphenylpropanoid metabolism, disease resistance (WO9813486-A1),expression of gibberellin-regulated genes (WO9700961-A1), expression ofstress-related genes (WO9916878-A1), active carbohydrate secretion andflavonol metabolism in antirrhinum flowers (Jackson et al. (1992) PlantCell 3(2):115-125). The first plant transcription activator genedescribed at the molecular level was the maize c1 gene which encodes aMyb protein (Paz-Ares et al. (1987) EMBO J 16:3553-3558) involved inregulating anthocyanin biosynthesis by trans-activating genes such asc2, A1 and Bz1 which encode enzymes involved in the pathway.

There is a great deal of interest in identifying the genes that encodeproteins involved in transcriptional regulation in plants. These genesmay be used in plant cells to control gene expression constitutively, inspecific tissues or at various times during development. Accordingly,the availability of nucleic acid sequences encoding all or a portion ofa Myb-related transcription factor would facilitate studies to betterunderstand gene regulation in plants and provide genetic tools toenhance or otherwise alter the expression of genes controlled byMyb-related transcription factors.

SUMMARY OF THE INVENTION

The present invention relates to isolated polynucleotides comprising anucleotide sequence encoding a first polypeptide of at least 50 aminoacids that has at least 80% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of a corn Myb-related transcription factor polypeptide of SEQID NO:2, a rice Myb-related transcription factor polypeptide of SEQ IDNO:12, and a wheat Myb-related transcription factor polypeptide of SEQID NO:56. The present invention also relates to isolated polynucleotidescomprising a nucleotide sequence encoding a first polypeptide of atleast 50 amino acids that has at least 85% identity based on the Clustalmethod of alignment when compared to a polypeptide selected from thegroup consisting of a corn Myb-related transcription factor polypeptideof SEQ ID NO:8, and a soybean Myb-related transcription factorpolypeptide of SEQ ID NO:28. The present invention also relates toisolated polynucleotides comprising a nucleotide sequence encoding afirst polypeptide of at least 50 amino acids that has at least 90%identity based on the Clustal method of alignment when compared to arice Myb-related transcription factor polypeptide of SEQ ID NO:16. Thepresent invention also relates to isolated polynucleotides comprising anucleotide sequence encoding a first polypeptide of at least 50 aminoacids that has at least 95% identity based on the Clustal method ofalignment when compared to a soybean Myb-related transcription factorpolypeptide of SEQ ID NO:52. The present invention also relates toisolated polynucleotides comprising a nucleotide sequence encoding afirst polypeptide of at least 100 amino acids that has at least 80%identity based on the Clustal method of alignment when compared to apolypeptide selected from the group consisting of a corn Myb-relatedtranscription factor polypeptide of SEQ ID NO:6, a rice Myb-relatedtranscription factor polypeptide of SEQ ID NO:14, a soybean Myb-relatedtranscription factor polypeptide of SEQ ID NO:50, and a wheatMyb-related transcription factor polypeptide of SEQ ID NO:58. Thepresent invention also relates to isolated polynucleotides comprising anucleotide sequence encoding a first polypeptide of at least 100 aminoacids that has at least 85% identity based on the Clustal method ofalignment when compared to a wheat Myb-related transcription factorpolypeptide of SEQ ID NO:60. The present invention also relates toisolated polynucleotides comprising a nucleotide sequence encoding afirst polypeptide of at least 100 amino acids that has at least 90%identity based on the Clustal method of alignment when compared to apolypeptide selected from the group consisting of a corn Myb-relatedtranscription factor polypeptide of SEQ ID NO:4, a corn Myb-relatedtranscription factor polypeptide of SEQ ID NO:10, a rice Myb-relatedtranscription factor polypeptide of SEQ ID NO:22, a rice Myb-relatedtranscription factor polypeptide of SEQ ID NO:24, and a wheatMyb-related transcription factor polypeptide of SEQ ID NO:62. Thepresent invention also relates to isolated polynucleotides comprising anucleotide sequence encoding a first polypeptide of at least 100 aminoacids that has at least 95% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of a rice Myb-related transcription factor polypeptide of SEQID NO:18 and a rice Myb-related transcription factor polypeptide of SEQID NO:20. The present invention also relates to isolated polynucleotidescomprising a nucleotide sequence encoding a first polypeptide of atleast 150 amino acids that has at least 80% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of a rice Myb-related transcription factorpolypeptide of SEQ ID NO:26, a soybean Myb-related transcription factorpolypeptide of SEQ ID NO:34, a soybean Myb-related transcription factorpolypeptide of SEQ ID NO:38, a soybean Myb-related transcription factorpolypeptide of SEQ ID NO:40, a soybean Myb-related transcription factorpolypeptide of SEQ ID NO:42, a soybean Myb-related transcription factorpolypeptide of SEQ ID NO:48, and a soybean Myb-related transcriptionfactor polypeptide of SEQ ID NO:54. The present invention also relatesto isolated polynucleotides comprising a nucleotide sequence encoding afirst polypeptide of at least 150 amino acids that has at least 85%identity based on the Clustal method of alignment when compared to apolypeptide selected from the group consisting of a soybean Myb-relatedtranscription factor polypeptide of SEQ ID NO:32, a soybean Myb-relatedtranscription factor polypeptide of SEQ ID NO:44, and a soybeanMyb-related transcription factor polypeptide of SEQ ID NO:46. Thepresent invention also relates to isolated polynucleotides comprising anucleotide sequence encoding a first polypeptide of at least 200 aminoacids that has at least 80% identity based on the Clustal method ofalignment when compared to a soybean Myb-related transcription factorpolypeptide of SEQ ID NO:36. The present invention also relates toisolated polynucleotides comprising a nucleotide sequence encoding afirst polypeptide of at least 200 amino acids that has at least 85%identity based on the Clustal method of alignment when compared to asoybean Myb-related transcription factor polypeptide of SEQ ID NO:30.The present invention also relates to an isolated polynucleotidecomprising the complement of the nucleotide sequences described above.

It is preferred that the isolated polynucleotides of the claimedinvention consist of a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and61 that codes for the polypeptide selected from the group consisting ofSEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, and 62. Thepresent invention also relates to an isolated polynucleotide comprisinga nucleotide sequences of at least one of 60 (preferably at least one of40, most preferably at one least of 30) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61 and thecomplement of such nucleotide sequences.

The present invention relates to a chimeric gene comprising an isolatedpolynucleotide of the present invention operably linked to suitableregulatory sequences.

The present invention relates to an isolated host cell comprising achimeric gene of the present invention or an isolated polynucleotide ofthe present invention. The host cell may be eukaryotic, such as a yeastor a plant cell, or prokaryotic, such as a bacterial cell. The presentinvention also relates to a virus, preferably a baculovirus, comprisingan isolated polynucleotide of the present invention or a chimeric geneof the present invention.

The present invention relates to a process for producing an isolatedhost cell comprising a chimeric gene of the present invention or anisolated polynucleotide of the present invention, the process comprisingeither transforming or transfecting an isolated compatible host cellwith a chimeric gene or isolated polynucleotide of the presentinvention.

The present invention relates to a Myb-related transcription factorpolypeptide of at least 50 amino acids that has at least 80% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of SEQ ID NOs:2, 12, and 56. Thepresent invention also relates to a Myb-related transcription factorpolypeptide of at least 50 amino acids that has at least 85% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of SEQ ID NOs:8 and 28. The presentinvention also relates to a Myb-related transcription factor polypeptideof at least 50 amino acids that has at least 90% identity based on theClustal method of alignment when compared to a rice Myb-relatedtranscription factor polypeptide of SEQ ID NO:16. The present inventionalso relates to a Myb-related transcription factor polypeptide of atleast 50 amino acids that has at least 95% identity based on the Clustalmethod of alignment when compared to a soybean Myb-related transcriptionfactor polypeptide of SEQ ID NO:52. The present invention also relatesto a Myb-related transcription factor polypeptide of at least 100 aminoacids that has at least 80% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NOs:6, 14, 50, and 58. The present invention alsorelates to a Myb-related transcription factor polypeptide of at least100 amino acids that has at least 85% identity based on the Clustalmethod of alignment when compared to a wheat Myb-related transcriptionfactor polypeptide of SEQ ID NO:60. The present invention also relates aMyb-related transcription factor polypeptide of at least 100 amino acidsthat has at least 90% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:4, 10, 22, 24, and 62. The present invention also relates to aMyb-related transcription factor polypeptide of at least 100 amino acidsthat has at least 95% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:18 and 20. The present invention also relates to a Myb-relatedtranscription factor polypeptide of at least 150 amino acids that has atleast 80% identity based on the Clustal method of alignment whencompared to a polypeptide selected from the group consisting of SEQ IDNOs:26, 34, 38, 40, 42, 48, and 54. The present invention also relatesto a Myb-related transcription factor polypeptide of at least 150 aminoacids that has at least 85% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NOs:32, 44, and 46. The present invention alsorelates to a Myb-related transcription factor polypeptide of at least200 amino acids that has at least 80% identity based on the Clustalmethod of alignment when compared to a soybean Myb-related transcriptionfactor polypeptide of SEQ ID NO:36. The present invention also relatesto a Myb-related transcription factor polypeptide of at least 200 aminoacids that has at least 85% identity based on the Clustal method ofalignment when compared to a soybean Myb-related transcription factorpolypeptide of SEQ ID NO:30.

The present invention relates to a method of selecting an isolatedpolynucleotide that affects the level of expression of a Myb-relatedtranscription factor polypeptide in a host cell, preferably a plantcell, the method comprising the steps of:

-   -   constructing an isolated polynucleotide of the present invention        or an isolated chimeric gene of the present invention;    -   introducing the isolated polynucleotide or the isolated chimeric        gene into a host cell;    -   measuring the level of a Myb-related transcription factor        polypeptide in the host cell containing the isolated        polynucleotide; and    -   comparing the level of a Myb-related transcription factor        polypeptide in the host cell containing the isolated        polynucleotide with the level of a Myb-related transcription        factor polypeptide in a host cell that does not contain the        isolated polynucleotide.

The present invention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of a Myb-related transcriptionfactor polypeptide gene, preferably a plant Myb-related transcriptionfactor polypeptide gene, comprising the steps of: synthesizing anoligonucleotide primer comprising a nucleotide sequence of at least oneof 60 (preferably at least one of 40, most preferably at least one of30) contiguous nucleotides derived from a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, and 61 and the complement of such nucleotide sequences; andamplifying a nucleic acid fragment (preferably a cDNA inserted in acloning vector) using the oligonucleotide primer. The amplified nucleicacid fragment preferably will encode all or a portion of a Myb-relatedtranscription factor amino acid sequence.

The present invention also relates to a method of obtaining a nucleicacid fragment encoding all or a substantial portion of the amino acidsequence encoding a Myb-related transcription factor polypeptidecomprising the steps of: probing a cDNA or genomic library with anisolated polynucleotide of the present invention; identifying a DNAclone that hybridizes with an isolated polynucleotide of the presentinvention; isolating the identified DNA clone; and sequencing the cDNAor genomic fragment that comprises the isolated DNA clone.

BRIEF DESCRIPTION OF THE DRAWING

The invention can be more fully understood from the following detaileddescription and the accompanying drawing which forms a part of thisapplication.

FIG. 1A-1G depicts the amino acid alignment between the Myb-relatedtranscription factor encoded by the nucleotide sequences derived fromcorn clone cta1n.pk0079.e9 (SEQ ID NO:10), contig assembled from riceclones rr1.pk0027.g9 and rr1.pk077.n9 (SEQ ID NO:14), rice cloner10n.pk082.c13 (SEQ ID NO:26), soybean clone sfl1.pk0032.g4 (SEQ IDNO:30), soybean clone sfl1.pk0086.a9 (SEQ ID NO:32), soybean clonesfl1.pk0091.a2 (SEQ ID NO:34), soybean clone sfl1.pk0091.a2 (SEQ IDNO:36), soybean clone sfl1.pk0003.a3 (SEQ ID NO:42), soybean clonesrr3c.pk002.k6 (SEQ ID NO:44), soybean clone ses9c.pk002.o16 (SEQ IDNO:46), soybean clone s12.pk127.e14 (SEQ ID NO:48), soybean clonesrc3c.pk010.122 (SEQ ID NO:50), soybean clone sgs4c.pk004.j24 (SEQ IDNO:54), and a Myb-related transcription factor-encoding nucleic acidfragment from Pisum sativum (NCBI General Identification No. 1841475)(SEQ ID NO:63) Amino acids which are conserved among all and at leasttwo sequences with an amino acid at that position are indicated with anasterisk (*) above them. Dashes are used by the program to maximizealignment of the sequences.

SEQUENCE DESCRIPTIONS

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. Table 1 also identifies the cDNA clonesas individual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), contigs assembled from twoor more ESTs (“Contig”), contigs assembled from an FIS and one or moreESTs (“Contig*”), or sequences encoding the entire protein derived froman FIS, a contig, or an FIS and PCR (“CGS”). Nucleotide SEQ ID NOs:7,11, 23, 27, 51, and 55 correspond to nucleotide SEQ ID NOs:1, 3, 9, 5,11, and 7, respectively, presented in U.S. Provisional Application No.60/110,609, filed Dec. 2, 1998. Amino acid SEQ ID NOs:8, 12, 24, 28, 52,and 56 correspond to amino acid SEQ ID NOs: 2, 4, 10, 6, 12, and 8,respectively presented in U.S. Provisional Application No. 60/110,609,filed Dec. 2, 1998. The sequence descriptions and Sequence Listingattached hereto comply with the rules governing nucleotide and/or aminoacid sequence disclosures in patent applications as set forth in 37C.F.R. §1.821-1.825.

TABLE 1 Myb-related Transcription Factors Clone SEQ ID NO: ProteinDesignation Status (Nucleotide) (Amino Acid) Myb-related TranscriptionFactor Contig of: Contig 1 2 (Corn) csi1n.pk0008.b5 csi1n.pk0028.h7Myb-related Transcription Factor Contig of: Contig 3 4 (Corn)p0008.cb3ld06r p0026.ccrbd36rb Myb-related Transcription Factorchpc8.pk0002.d9 EST 5 6 (Corn) Myb-related Transcription Factorcta1n.pk0079.e9 EST 7 8 (Corn) Myb-related Transcription Factorcta1n.pk0079.e9 CGS 9 10 (Corn) Myb-related Transcription Factorrr1.pk077.n9 EST 11 12 (Rice) Myb-related Transcription Factor Contigof: CGS 13 14 (Rice) rr1.pk0027.g9 rr1.pk077.n9 Myb-relatedTranscription Factor rr1.pk088.p6 EST 15 16 (Rice) Myb-relatedTranscription Factor rr1.pk0037.g7 EST 17 18 (Rice) Myb-relatedTranscription Factor rds3c.pk002.c6 EST 19 20 (Rice) Myb-relatedTranscription Factor Contig of: Contig 21 22 (Rice) rlr24.pk0090.f5rlr48.pk0012.c11 Myb-related Transcription Factor rl0n.pk082.c13 EST 2324 (Rice) Myb-related Transcription Factor rl0n.pk082.c13 CGS 25 26(Rice) Myb-related Transcription Factor sfl1.pk0032.g4 EST 27 28(Soybean) Myb-related Transcription Factor sfl1.pk0032.g4 CGS 29 30(Soybean) Myb-related Transcription Factor sfl1.pk0086.a9 CGS 31 32(Soybean) Myb-related Transcription Factor sfl1.pk0091.a2 CGS 33 34(Soybean) Myb-related Transcription Factor sfl1.pk0105.e6 CGS 35 36(Soybean) Myb-related Transcription Factor sfl1.pk125.p19 FIS 37 38(Soybean) Myb-related Transcription Factor se6.pk0048.a12 FIS 39 40(Soybean) Myb-related Transcription Factor sfl1.pk0003.a3 CGS 41 42(Soybean) Myb-related Transcription Factor srr3c.pk002.k6 CGS 43 44(Soybean) Myb-related Transcription Factor ses9c.pk002.o16 CGS 45 46(Soybean) Myb-related Transcription Factor sl2.pk127.e14 CGS 47 48(Soybean) Myb-related Transcription Factor src3c.pk010.i22 CGS 49 50(Soybean) Myb-related Transcription Factor sgs4c.pk004.j24 EST 51 52(Soybean) Myb-related Transcription Factor sgs4c.pk004.j24 CGS 53 54(Soybean) Myb-related Transcription Factor wr1.pk0139.g11 EST 55 56(Wheat) Myb-related Transcription Factor wr1.pk0139.g11 FIS 57 58(Wheat) Myb-related Transcription Factor wdk3c.pk006.n12 EST 59 60(Wheat) Myb-related Transcription Factor wlm1.pk0027.a5 EST 61 62(Wheat)

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “polynucleotide” is a nucleotide sequence such as anucleic acid fragment. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least one of 60contiguous nucleotides, preferably at least one of 40 contiguousnucleotides, most preferably one of at least 30 contiguous nucleotides,of the nucleic acid sequence of the SEQ ID NOs:1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, or the complement of such sequences.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 30 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least one of 60 (preferably at least one of40, most preferably at least one of 30) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61 and thecomplement of such nucleotide sequences may be used in methods ofselecting an isolated polynucleotide that affects the expression of apolypeptide in a plant cell. A method of selecting an isolatedpolynucleotide that affects the level of expression of a polypeptide(such as a Myb-related transcription factor) in a host cell (eukaryotic,such as plant or yeast, prokaryotic such as bacterial, or viral) maycomprise the steps of: constructing an isolated polynucleotide of thepresent invention or an isolated chimeric gene of the present invention;introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell; measuring the level of a polypeptide in the host cellcontaining the isolated polynucleotide; and comparing the level of apolypeptide in the host cell containing the isolated polynucleotide withthe level of a polypeptide in a host cell that does not contain theisolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least about 70%identical, most preferably at least about 80% identical to the aminoacid sequences reported herein. Preferred nucleic acid fragments encodeamino acid sequences that are at least about 85% identical to the aminoacid sequences reported herein. More preferred nucleic acid fragmentsencode amino acid sequences that are at least about 90% identical to theamino acid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are at least about 95%identical to the amino acid sequences reported herein. Suitable nucleicacid fragments not only have the above homologies but typically encode apolypeptide having at least about 50 amino acids, preferably at leastabout 100 amino acids, more preferably at least about 150 amino acids,still more preferably at least about 200 amino acids, and mostpreferably at least about 250 amino acids. Sequence alignments andpercent identity calculations were performed using the Megalign programof the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison,Wis.). Multiple alignment of the sequences was performed using theClustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).Default parameters for pairwise alignments using the Clustal method wereKTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Underexpression”refers to the production of a gene product in transgenic organisms atlevels below that of levels of production in normal or non-transformedorganisms. “Co-suppression” refers to the production of sense RNAtranscripts capable of suppressing the expression of identical orsubstantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of severalMyb-related transcription factors have been isolated and identified bycomparison of random plant cDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

For example, genes encoding other Myb-related transcription factors,either as cDNAs or genomic DNAs, could be isolated directly by using allor a portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast one of 60 (preferably one of at least 40, most preferably one ofat least 30) contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, and 61 and the complement of such nucleotidesequences may be used in such methods to obtain a nucleic acid fragmentencoding a substantial portion of an amino acid sequence of apolypeptide (such as a Myb-related transcription factor. The presentinvention relates to a method of obtaining a nucleic acid fragmentencoding a substantial portion of a polypeptide of a gene (such asMyb-related transcription factor) preferably a substantial portion of aplant polypeptide of a gene, comprising the steps of: synthesizing anoligonucleotide primer comprising a nucleotide sequence of at least oneof 60 (preferably at least one of 40, most preferably at least one of30) contiguous nucleotides derived from a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, and 61 and the complement of such nucleotide sequences; andamplifying a nucleic acid fragment (preferably a cDNA inserted in acloning vector) using the oligonucleotide primer. The amplified nucleicacid fragment preferably will encode a portion of a polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed polypeptides are presentat higher or lower levels than normal or in cell types or developmentalstages in which they are not normally found. This would have the effectof altering the level of expression of Myb-regulated genes in thosecells, and consequently the phenotype affected by those Myb-regulatedgenes.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can then beconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds, and is not an inherentpart of the invention. For example, one can screen by looking forchanges in gene expression by using antibodies specific for the proteinencoded by the gene being suppressed, or one could establish assays thatspecifically measure enzyme activity. A preferred method will be onewhich allows large numbers of samples to be processed rapidly, since itwill be expected that a large number of transformants will be negativefor the desired phenotype.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded Myb-related transcription factors. An example of a vectorfor high level expression of the instant polypeptides in a bacterialhost is provided (Example 6).

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci. USA 86:9402-9406; Koes et al.(1995) Proc. Natl. Acad. Sci. USA 92:8149-8153; Bensen et al. (1995)Plant Cell 7:75-84). The latter approach may be accomplished in twoways. First, short segments of the instant nucleic acid fragments may beused in polymerase chain reaction protocols in conjunction with amutation tag sequence primer on DNAs prepared from a population ofplants in which Mutator transposons or some other mutation-causing DNAelement has been introduced (see Bensen, supra). The amplification of aspecific DNA fragment with these primers indicates the insertion of themutation tag element in or near the plant gene encoding the instantpolypeptide. Alternatively, the instant nucleic acid fragment may beused as a hybridization probe against PCR amplification productsgenerated from the mutation population using the mutation tag sequenceprimer in conjunction with an arbitrary genomic site primer, such asthat for a restriction enzyme site-anchored synthetic adaptor. Witheither method, a plant containing a mutation in the endogenous geneencoding the instant polypeptide can be identified and obtained. Thismutant plant can then be used to determine or confirm the naturalfunction of the instant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various corn, rice, soybean andwheat tissues were prepared. The characteristics of the libraries aredescribed below.

TABLE 2 cDNA Libraries from Corn, Rice, Soybean and Wheat Library TissueClone chpc8 Corn (Zea mays L.) (MBS847) 8 Day Old Shoot Treatedchpc8.pk0002.d9 8 Hours With PDO Herbicide MK593* csi1n Corn (Zea maysL.) Silk** csi1n.pk0008.b5 csi1n.pk0028.h7 cta1n Corn (Zea mays L.)Tassel** cta1n.pk0079.e9 p0008 Corn (Zea mays L.) 3 Week Old Leafp0008.cb3ld06r p0026 Corn (Zea mays L.) Regenerating Callus (Hi-II 223aand p0026.ccrbd36rb 1129e), 5 Days After Auxin Removal rds3c Rice (Oryzasativa) Developing Seed From Top of the Plant rds3c.pk002.c6 rl0n Rice(Oryza sativa) 15 Day Old Leaf** rl0n.pk082.c13 rlr24 Resistant Rice(Oryza sativa) Leaf 15 Days After rlr24.pk0090.f5 Germination, 24 HoursAfter Infection of Strain Magnaporthe grisea 4360-R-62 (AVR2-YAMO) rlr48Resistant Rice (Oryza sativa) Leaf 15 Days After rlr48.pk0012.c11Germination, 48 Hours After Infection of Strain Magnaporthe grisea4360-R-62 (AVR2-YAMO) rr1 Rice (Oryza sativa) Root of Two Week OldDeveloping rr1.pk0027.g9 Seedling rr1.pk0037.g7 rr1.pk077.n9rr1.pk088.p6 se6 Soybean (Glycine max L) Embryo, 26 Days After Floweringse6.pk0048.a12 ses9c Soybean (Glycine max L) Embryogenic Suspensionses9c.pk002.o16 sfl1 Soybean (Glycine max L) Immature Flowersfl1.pk0003.a3 sfl1.pk0032.g4 sfl1.pk0086.a9 sfl1.pk0091.a2sfl1.pk0105.e6 sfl1.pk125.p19 sgs4c Soybean (Glycine max L) Seed 2 DaysAfter Germination sgs4c.pk004.j24 sl2 Soybean (Glycine max L)Two-Week-Old Developing sl2.pk127.e14 Seedling Treated With 2.5 ppmchlorimuron src3c Soybean (Glycine max L) 8 Day Old Root Infected Withsrc3c.pk010.i22 Cyst Nematode srr3c Soybean (Glycine max L) 8 Day OldRoot srr3c.pk002.k6 wdk3c Wheat (Triticum aestivum L) Developing Kernel,wdk3c.pk006.n12 14 Days After Anthesis wlm1 Wheat (Triticum aestivum L)Seedling 1 Hour After wlm1.pk0027.a5 Inoculation With Erysiphe graminisf. sp tritici wr1 Wheat (Triticum aestivum L) Root From 7 Day Oldwr1.pk0139.g11 Seedling Light Grown *Application of2-[(2,4-dihydro-2,6,9-trimethyl[1]benzothiopyrano[4,3-c]pyrazol-8-yl)carbonyl]-1,3-cyclohexanedioneS,S-dioxide; synthesis and methods of using this compound are describedin WO 97/19087, incorporated herein by reference. **These libraries werenormalized essentially as described in U.S. Pat. No. 5,482,845,incorporated herein by reference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Determination of complete nucleotide sequence of cDNA inserts may beaccomplished by a number of methods well-known to those skilled in theart (Maniatis). For example, this may be accomplished stepwise, whereinoligonucleotides near the 5′ or 3′ end of the sequence may besynthesized which can then serve as primers for sequencing reactionsthat will extend the known sequence. Another set of oligonucleotidesnear the 5′ or 3′ end of the new sequence in the next round primeanother set of sequencing reactions to obtain more sequence information.These steps are repeated until the complete nucleotide sequence isdetermined.

Example 2 Identification of cDNA Clones

cDNA clones encoding Myb-related transcription factors were identifiedby conducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/)searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) Nat.Genet. 3:266-272) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Myb-RelatedTranscription Factors

The BLASTX search using the EST sequence from clone cta1n.pk0079.e9revealed similarity of the protein encoded by the cDNA to a Myb-relatedtranscription factor from Craterostigma plantagineum (NCBI IdentifierNo. gi 1002800). The BLASTX search using the EST sequence from clonerr1.pk077.n9 revealed similarity of the protein encoded by the cDNA to aMyb-related transcription factor from Arabidopsis thaliana (NCBIIdentifier No. gi 1732513). The BLASTX search using the EST sequencefrom clone sfl1.pk0032.g4 revealed similarity of the protein encoded bythe cDNA to a Myb-related transcription factor from Pisum sativum (NCBIIdentifier No. gi 1841475). The BLASTX search using the EST sequencefrom clone wr1.pk0139.g11 revealed similarity of the protein encoded bythe cDNA to a Myb-related transcription factor from Arabidopsis thaliana(NCBI Identifier No. gi 2832500). The BLAST results for each of theseESTs are shown in Table 3:

TABLE 3 BLAST Results forClones Encoding Polypeptides Homologous toPlant Myb-related Transcription Factors Clone BLAST pLog Scorecta1n.pk0079.e9 39.00 rr1.pk077.n9 27.70 sfl1.pk0032.g4 38.50wr1.pk0139.g11 16.00

The sequence of a portion of the cDNA insert from clone cta1n.pk0079.e9is shown in SEQ ID NO:7; the deduced amino acid sequence of this cDNA isshown in SEQ ID NO:8. The sequence of a portion of the cDNA insert fromclone rr1.pk077.n9 is shown in SEQ ID NO:11; the deduced amino acidsequence of this cDNA is shown in SEQ ID NO:12. The sequence of aportion of the cDNA insert from clone sfl1.pk0032.g4 is shown in SEQ IDNO:27; the deduced amino acid sequence of this cDNA is shown in SEQ IDNO:28. The sequence of a portion of the cDNA insert from clonewr1.pk0139.g11 is shown in SEQ ID NO:55; the deduced amino acid sequenceof this cDNA is shown in SEQ ID NO:56. BLAST scores and probabilitiesindicate that the instant nucleic acid fragments encode portions of aMyb-related transcription factor protein.

The BLASTX search using the EST sequences from clones r10n.pk082.c13 andsgs4c.pk004.j24 revealed similarity of the proteins encoded by the cDNAsto a Myb-related transcription factor protein from Pisum sativum (NCBIIdentifier No. gi 82307). The BLAST results for each of these ESTs areshown in Table 4:

TABLE 4 BLAST Results for Clones Encoding Polypeptides Homologous toPlant Myb-related Transcription Factors Clone BLAST pLog Scorerl0n.pk082.c13 62.50 sgs4c.pk004.j24 47.50

The sequence of a portion of the cDNA insert from clone r10n.pk082.c13is shown in SEQ ID NO:23; the deduced amino acid sequence of this cDNAis shown in SEQ ID NO:24. The sequence of a portion of the cDNA insertfrom clone sgs4c.pk004.j24 is shown in SEQ ID NO:51; the deduced aminoacid sequence of this cDNA is shown in SEQ ID NO:52. BLAST scores andprobabilities indicate that the instant nucleic acid fragments encodeportions of a Myb-related transcription factor protein.

The BLASTX search using the sequences from clones listed in Table 5revealed similarity of the polypeptides encoded by the cDNAs toMyb-related transcription factors from different plant species includingPisum sativum (NCBI General Identification No. 1841475), Arabidopsisthaliana (NCBI General Identification Nos. 3941480 and 3941528),Craterostigma plantagineum (NCBI General Identification Nos. 1002796,1002798, and 1002800), and Antirrhinum majus (NCBI GeneralIdentification No. 82307). Shown in Table 5 are the BLAST results forindividual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), contigs assembled from twoor more ESTs (“Contig”), contigs assembled from an FIS and one or moreESTs (“Contig*”), or sequences encoding the entire protein derived froman FIS, a contig, or an FIS and PCR (“CGS”):

TABLE 5 BLAST Results for Sequences Encoding Polypeptides Homologous toMyb-related Transcription Factors BLAST Results NCBI General CloneStatus Identification No. pLog Score Contig of: Contig 1841475 54.40csi1n.pk0008.b5 csi1n.pk0028.h7 Contig of: Contig 3941480 70.10p0008.cb3ld06r p0026.ccrbd36rb chpc8.pk0002.d9 EST 1002800 44.70cta1n.pk0079.e9 CGS 1002800 68.52 Contig of: CGS 3941480 50.00rr1.pk0027.g9 rr1.pk077.n9 rr1.pk088.p6 EST 3941480 22.52 rr1.pk0037.g7EST 1002798 79.70 rds3c.pk002.c6 EST 1002798 62.00 Contig of: Contig1002800 55.04 rlr24.pk0090.f5 rlr48.pk0012.c11 rl0n.pk082.c13 CGS 8230784.22 sfl1.pk0032.g4 CGS 1841475 96.52 sfl1.pk0086.a9 CGS 1841475 92.22sfl1.pk0091.a2 CGS 1841475 75.10 sfl1.pk0105.e6 CGS 1841475 96.40sfl1.pk125.p19 FIS 1841475 90.52 se6.pk0048.a12 FIS 1002798 66.70sfl1.pk0003.a3 CGS 1002796 59.70 srr3c.pk002.k6 CGS 1002798 77.40ses9c.pk002.o16 CGS 1002798 73.70 sl2.pk127.e14 CGS 1002800 60.70src3c.pk010.i22 CGS 1002800 57.05 sgs4c.pk004.j24 CGS 82307 90.52wr1.pk0139.g11 FIS 3941480 48.52 wdk3c.pk006.n12 EST 1002796 43.70wlm1.pk0027.a5 EST 3941528 73.70

FIG. 1 presents an alignment of the amino acid sequences set forth inSEQ ID NOs:10, 14, 26, 30, 32, 34, 36, 42, 44, 46, 48, 50, and 54 andthe Pisum sativum sequence (NCBI General Identification No. 1841475; SEQID NO:63). The data in Table 6 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs:10, 14, 26,30, 32, 34, 36, 42, 44, 46, 48, 50, and 54 and the Pisum sativumsequence (NCBI General Identification No. 1841475; SEQ ID NO:63).

TABLE 6 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toMyb-related Transcription Factors Percent Identity to NCBI GeneralIdentification SEQ ID NO. No. 1841475 10 43.8 14 33.2 26 30.9 30 75.6 3274.1 34 64.1 36 77.7 42 43.8 44 48.5 46 46.5 48 45.6 50 46.1 54 31.8

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode all or a substantial portion of a Myb-related transcriptionfactor. These sequences represent the first soybean and wheat sequencesencoding Myb-related transcription factors. Nucleic acid fragmentsencoding Myb-related transcription factors have previously been isolatedfrom rice and corn (Marocco et al. (1989) Mol Gen Genet. 216:183-187;Pandolfi et al. (1997) Plant Physiol 114:747).

Example 4 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptide insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptide, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μl of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μl ofa 2.5 M solution) and spermidine free base (20 μl of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μl of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μl of ethanol. Analiquot (5 μl) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptide and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μl of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptide are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μl of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 7 Assaying Myb-Related Transcription Factor Activity

The polypeptides described herein may be produced using any number ofmethods known to those skilled in the art. Such methods include, but arenot limited to, expression in bacteria as described in Example 6, orexpression in eukaryotic cell culture, in planta, and using viralexpression systems in suitably infected organisms or cell lines. Theinstant −4B. In an alternate embodiment, a thioredoxin fusion proteinmay be eluted using dithiothreitol; however, elution may be accomplishedusing other reagents which interact to displace the thioredoxin from theresin. These reagents include β-mercaptoethanol or other reduced thiol.The eluted fusion protein may be subjected to further purification bypolypeptides may be expressed either as mature forms of the proteins asobserved in vivo or as fusion proteins by covalent attachment to avariety of enzymes, proteins or affinity tags. Common fusion proteinpartners include glutathione S-transferase (“GST”), thioredoxin (“Trx”),maltose binding protein, and C- and/or N-terminal hexahistidinepolypeptide (“(His)₆”). The fusion proteins may be engineered with aprotease recognition site at the fusion point so that fusion partnerscan be separated by protease digestion to yield intact mature enzyme.Examples of such proteases include thrombin, enterokinase and factor Xa.However, any protease can be used which specifically cleaves the peptideconnecting the fusion protein and the enzyme.

Purification of the instant polypeptides, if desired, may utilize anynumber of separation technologies familiar to those skilled in the artof protein purification. Examples of such methods include, but are notlimited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptides may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharosetraditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond™ affinityresin or other resin.

Crude, partially purified or purified enzyme, either alone or as afusion protein, may be utilized in assays to verify over- orunderexpression of functional Myb-related transcription factor proteinin transgenic plants and transformed bacterial cells. Assays may beconducted under well known experimental conditions which permit optimalenzymatic activity. For example, assays for Myb-related transcriptionfactors are presented by Moyano et al. (1996) Plant Cell 8:1519-1532.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

The disclosure of each reference set forth above is incorporated hereinby reference in its entirety.

What is claimed is:
 1. An isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: (a) a firstnucleotide sequence encoding a polypeptide of at least 50 amino acidsthat has at least 80% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NO: 2, SEQ ID NO: 12, and SEQ ID NO:56; (b) a second nucleotidesequence encoding a polypeptide of at least 50 amino acids that has atleast 85% identity based on the Clustal method of alignment whencompared to a polypeptide selected from the group consisting of SEQ IDNO: 8 and SEQ ID NO:28; (c) a third nucleotide sequence encoding apolypeptide of at least 50 amino acids that has at least 90% identitybased on the Clustal method of alignment when compared to a polypeptideof SEQ ID NO: 16; (d) a fourth nucleotide sequence encoding apolypeptide of at least 50 amino acids that has at least 95% identitybased on the Clustal method of alignment when compared to polypeptide ofSEQ ID NO: 52; (e) a fifth nucleotide sequence encoding a polypeptide ofat least 100 amino acids that has at least 80% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO:50, andSEQ ID NO:58; (f) a sixth nucleotide sequence encoding a polypeptide ofat least 100 amino acids that has at least 85% identity based on theClustal method of alignment when compared to SEQ ID NO: 60; (g) aseventh nucleotide sequence encoding a polypeptide of at least 100 aminoacids that has at least 90% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:24,and SEQ ID NO:62; (h) a eighth nucleotide sequence encoding apolypeptide of at least 100 amino acids that has at least 95% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of SEQ ID NO: 18 and SEQ ID NO:20;(i) a ninth nucleotide sequence encoding a polypeptide of at least 150amino acids that has at least 80% identity based on the Clustal methodof alignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO:38, SEQ ID NO:40,SEQ ID NO:42, SEQ ID NO:48, and SEQ ID NO:54; (j) a tenth nucleotidesequence encoding a polypeptide of at least 150 amino acids that has atleast 85% identity based on the Clustal method of alignment whencompared to a polypeptide selected from the group consisting of SEQ IDNO: 32, SEQ ID NO:44, and SEQ ID NO:46; (k) an eleventh nucleotidesequence encoding a polypeptide of at least 200 amino acids that has atleast 80% identity based on the Clustal method of alignment whencompared to SEQ ID NO: 36; (l) a twelfth nucleotide sequence encoding apolypeptide of at least 200 amino acids that has at least 85% identitybased on the Clustal method of alignment when compared to SEQ ID NO: 30;and (m) a thirteenth nucleotide sequence comprising the complement of(a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), or (l).
 2. Theisolated polynucleotide of claim 1, wherein the isolated nucleotidesequence consists of a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and61 that codes for the polypeptide selected from the group consisting ofSEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, and
 62. 3. Theisolated polynucleotide of claim 1 wherein the nucleotide sequences areDNA.
 4. The isolated polynucleotide of claim 1 wherein the nucleotidesequences are RNA.
 5. A chimeric gene comprising the isolatedpolynucleotide of claim 1 operably linked to suitable regulatorysequences.
 6. An isolated host cell comprising the chimeric gene ofclaim
 5. 7. An isolated host cell comprising an isolated polynucleotideof claim 1 or claim
 3. 8. The isolated host cell of claim 7 wherein theisolated host is selected from the group consisting of yeast, bacteria,plant, and virus.
 9. A virus comprising the isolated polynucleotide ofclaim
 1. 10. A polypeptide selected from the group consisting of: (a) afirst sequence of at least 50 amino acids that has at least 80% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 12, andSEQ ID NO:56; (b) a second sequence of at least 50 amino acids that hasat least 85% identity based on the Clustal method of alignment whencompared to a polypeptide selected from the group consisting of SEQ IDNO: 8 and SEQ ID NO:28; (c) a third sequence of at least 50 amino acidsthat has at least 90% identity based on the Clustal method of alignmentwhen compared to SEQ ID NO: 16; (d) a fourth sequence of at least 50amino acids that has at least 95% identity based on the Clustal methodof alignment when compared to SEQ ID NO: 52; (e) a fifth sequence of atleast 100 amino acids that has at least 80% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO:50, andSEQ ID NO:58; (f) a sixth sequence of at least 100 amino acids that hasat least 85% identity based on the Clustal method of alignment whencompared to SEQ ID NO: 60; (g) a seventh sequence of at least 100 aminoacids that has at least 90% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:24,and SEQ ID NO:62; (h) an eighth sequence of at least 100 amino acidsthat has at least 95% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NO: 18 and SEQ ID NO:20; (i) a ninth sequence of at least 150 aminoacids that has at least 80% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO:38, SEQ ID NO:40,SEQ ID NO:42, SEQ ID NO:48, and SEQ ID NO:54; (j) a tenth sequence of atleast 150 amino acids that has at least 85% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of SEQ ID NO: 32, SEQ ID NO:44, and SEQ ID NO:46;(k) an eleventh sequence of at least 200 amino acids that has at least80% identity based on the Clustal method of alignment when compared toSEQ ID NO: 36; and (l) a twelfth sequence of at least 200 amino acidsthat has at least 85% identity based on the Clustal method of alignmentwhen compared to SEQ ID NO:
 30. 11. A method of selecting an isolatedpolynucleotide that affects the level of expression of a Myb-relatedtranscription factor polypeptide in a plant cell, the method comprisingthe steps of: (a) constructing an isolated polynucleotide comprising anucleotide sequence of at least one of 30 contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61 and thecomplement of such nucleotide sequences; (b) introducing the isolatedpolynucleotide into a plant cell; and (c) measuring the level of apolypeptide in the plant cell containing the polynucleotide to provide apositive selection means.
 12. The method of claim 11 wherein theisolated polynucleotide consists of a nucleotide sequence selected fromthe group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, and 61 that codes for the polypeptide selected from the groupconsisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, and62.
 13. A method of selecting an isolated polynucleotide that affectsthe level of expression of a Myb-related transcription factorpolypeptide in a plant cell, the method comprising the steps of: (a)constructing an isolated polynucleotide of claim 1; (b) introducing theisolated polynucleotide into a plant cell; and (c) measuring the levelof polypeptide in the plant cell containing the polynucleotide toprovide a positive selection means.
 14. A method of obtaining a nucleicacid fragment encoding a Myb-related transcription factor polypeptidecomprising the steps of: (a) synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least one of 30 contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and61 and the complement of such nucleotide sequences; and (b) amplifying anucleic acid sequence using the oligonucleotide primer.
 15. A method ofobtaining a nucleic acid fragment encoding a Myb-related transcriptionfactor polypeptide comprising the steps of: (a) probing a cDNA orgenomic library with an isolated polynucleotide comprising a nucleotidesequence of at least one of 30 contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61 and the complement ofsuch nucleotide sequences; (b) identifying a DNA clone that hybridizeswith the isolated polynucleotide; (c) isolating the identified DNAclone; and (d) sequencing the cDNA or genomic fragment that comprisesthe isolated DNA clone.
 16. An isolated polynucleotide comprising atleast one of 30 nucleotides derived from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, and the complementof such sequences.